Approximately 3 million tuberculosis-related deaths occur in the world annually and it is estimated that one third of the world's population is infected with M. tuberculosis (MTB) (Dye et al 1999). The rapid and accurate detection of the infection is crucial for the global control of the disease. At present, the diagnosis of active tuberculosis disease, and therefore the subsequent evaluation of the efficacy of anti-tuberculosis treatment, are based on the time-consuming microbiological culture of M. tuberculosis (that has 85% sensitivity), with a turnaround time of 26 weeks. This molecular diagnostic techniques proved to be useful only for culture-positive samples (American Thoracic Society 1997), and are available in highly specialized laboratories only. Therefore tuberculosis remains a global health problem because of the long time needed for microbiological diagnosis and because of the difficulty in monitoring the efficacy of anti-tuberculosis therapy. In the field, the tuberculin skin test (TST) is presently used to support the diagnosis of tuberculosis in patients without microbiological confirmation of the disease (Fine et al 1999, Huebner et al 1993, American Thoracic Society 2000, CDC MMWR Recommendation and Reports 1997). However, this test has significant limitations as purified protein derivative (PPD) used for TST is a crude precipitate of filtered MTB containing more than 200 antigens widely shared among environmental mycobacteria (Fine et al 1999, Huebner et al 1993, American Thoracic Society 2000. CDC MMWR Recommendation and Reports 1997). In patients with tuberculosis, the TST is 75-90% sensitive, but among those with disseminated disease, this sensitivity falls to 50% (Fine et al 1999, Huebner et al 1993, American Thoracic Society 2000). It is even lower in HIV+ patients with only mild degrees of immunosuppression (Fine et al 1999, Huebner et al 1993, American Thoracic Society 2000, CDC MMWR Recommendation and Reports 1997). Moreover, a positive TST does not discriminate between latent and active tuberculosis and thus, in subjects with a positive test, further clinical work is needed to obtain a correct diagnosis (Fine et al 1999, Huebner et al 1993).
From the immunological point of view, it has been demonstrated that M. tuberculosis evokes a strong cell-mediated immune response in vitro, with a high production of interferon (IFN)-gamma. The detection of T cells secreting this cytokine in response to M. tuberculosis antigens represents a tool to diagnose infection (Andersen et al 1993).
Recent studies have led to the identification of the genomic segment RD1, present In the M. tuberculosis complex but absent from M. bovis of BCG (Bacillus Calmette and Guerin), which is currently used as a vaccine and from the majority of environmental mycobacteria (Mahairas et al 1996, Harboe et al 1996, WO 2004/099771, EP 1 350 839 A: van Pinxteren 2000; Cockle 2002; Andersen 2001; WO 99/04005; WO 99/42076 A; WO 97/09428 A; WO 01/04151 A; WO 01/79274 A; WO 03/093307 A). RD1 encodes the Earty Secretory Antigenic Target (ESAT)-6 and the Culture Filtrate Protein (CFP)-10. These proteins, synthesized by the bacterium during the active replication phase, are dominant targets for cell-mediated immunity in animal models (Andersen et al 1995, Pollock et al 1997, WO 2004/099771, WO 00/26248 A; EP 1 350 839 A: van Pinxteren 2000; Cockle 2002; Andersen 2001; WO 99/04005; WO 99/42076 A; WO 97/09428 A; WO 01104151 A; WO 01/79274 A; WO 03/093307 A) and in patients with tuberculosis (Ulrichs et al 1998, Ravn et al 1999. WO 00/26248 A) and subjects exposed to M. tuberculosis who develop the disease within 2 years (Doherty et al 2002). For these reasons, the response of peripheral blood mononuclear cells (PBMC) to ESAT-6 or CFP-10 proteins (Lalvani et al 2001-a, 2001-b, and 2001-c, Pathan et al 2001) has been correlated with tuberculosis diagnosis by using the IFN-gamma ELISPOT assay. This technique allows estimating the frequency of T cells that are producing a given cytokine (for example, IFN-gamma) in response to a specific antigenic stimulus, for example ESAT-6, CFP-10, PPD (Purified Protein Derivative), etc. The response to the whole ESAT-8 or CFP-10 proteins allows discrimination between BCG-vaccinated subjects and subjects infected with M. tuberculosis. In fact, only the subjects who have been exposed to M. tuberculosis, either BCG-vaccinated or not, display an in vitro response to these proteins that are absent from M. bovis and BCG. In line with these studies two commercial tests (QuantiFERON-TB Gold—Cellestis Limited, Carnagie, Victoria, Australia and the T SPOT-TB, Oxford Immunotec, Oxford, UK) (Pai et al 2004, Mori et al 2004) were developed. Both tests evaluate cell mediated immune response to M. tuberculosis by measuring IFN-gamma released from T cells in response to M. tuberculosis antigens, using methods such as ELISA and ELISPOT respectively. In both assays M. tuberculosis antigens consist in ESAT-6 and CFP-10 overlapping peptides spanning the whole proteins. These assays appear to provide a quite accurate diagnosis of tuberculosis infection. However, such response does not allow discriminating between subjects with active tuberculosis and subjects with latent infection.
Thus, as in the case of a positivity to TST, a positive response to these immune tests based on RD1 overlapping peptides require further clinical workup to rule out an active tuberculosis. This example is particular relevant considering a subject with a suspect of active tuberculosis (thus with clinical symptoms of active disease) resulting positive to these conventional tuberculosis assays because with a latent TB infection (Richeldi et al, 2004a&b; Ravn, 2004; Brock, 2004). These results can lead to an incorrect diagnosis, because the subject may have a disease that mimics active tuberculosis, but different from tuberculosis (such as a bacterial pneumonia, a viral fever, a bronchitis) and thus he may start an incorrect treatment, he may be isolated from the community if the suspect is a pulmonary tuberculosis, with relevant human and economic costs.
Thus the distinction between active and latent tuberculosis is important to:                a) provide a correct diagnosis, because the active form of the disease leads to organ destruction and/or death and to the spread of infection the community; in contrast the latent infection does not involve organ destruction and it is not dangerous for the community        b) provide a correct and efficacious therapy, because active tuberculosis requires regime of first 2 months therapy with 4 drugs and then followed by 4 months therapy with 2 drugs, whereas for latent tuberculosis it is necessary to treat the patient with one drug for 6 months        c) save human and economic costs avoiding complex evaluations (i.e. clinical, radiological and surgery procedures).        
Our assay is different because first of all, we may discriminate between active and latent tuberculosis compared to other patent applications and scientific articles (US2004058399; WO02059605; WO0104151; WO0026248; WO9221097; WO 2004/099771, EP 1 350 839 A; van Pinxteren 2000; Cockle 2002; Andersen 2001; WO 99/04005; WO 99/42076 A; WO 97/09428 A; WO 01/04151 A; WO 01/79274 A: WO 03/093307 A). In addition we may monitor the therapy efficacy or failure in a more accurate way. In fact having set up a cut-off, in the case of therapy efficacy, the selected peptides response goes under the detection level, differently from others in which it is mentioned only a vaguely decrease of the response (Lalvani et al, 2001b; (US2004058399; WO 2004/099771, EP 1 350 839 A; van Pinxteren 2000; Cockle 2002; Andersen 2001; WO 99/04005; WO 99142076 A; WO 97/09428 A; WO 01/04151 A; WO 01/79274 A; WO 03/093307 A).
The possibility to monitor therapy efficacy by our assay is particularly relevant because it is:    a) easy to perform by a simple blood draw instead of a invasive surgery procedures;    b) unexpensive, compared to the surgery procedures involved in a biopsy, if an extra-pulmonary tuberculosis is suspected, or a broncoscopy, if the patient is sputum negative    c) more accurate compared to the assays based on RD1 overlapping peptide because our assay may distinguish between active and latent tuberculosis while the other immune assays are associated to tuberculosis infection (see below).